Project Summary Project Title: Development of sensitive fluorescent probes for physiological Zn2+ over large concentration ranges Principal Investigator: Lei Zhu Co-Principal Investigator: Michael W. Davidson Co-Principal Investigator: Cathy W. Levenson We aim to develop fluorescent probes for imaging free zinc ions (Zn2+) in physiological settings with both high sensitivity and large concentration coverage. Zn2+ is known to play structural, catalytic, and other roles in human physiology. The disruption of Zn2+ homeostasis is pathological. The exact functions of Zn2+ in different biochemical pathways are not completely elucidated, partly due to the lack of tools to accurately determine the distribution and movement of Zn2+ in heterogeneous and dynamic biological media. One particular challenge that distinguishes Zn2+ from other physiologically significant substances is the more than six orders of magnitude concentration range of Zn2+ in biological systems. The basal level of free Zn2+ (Zn2+ not tightly bound with proteins) in mammalian cells is believed to be between picomolar and nanomolar. However, elevated Zn2+ concentrations approaching millimolar have been often found in certain specialized cells such as brain neurons and certain subcellular organelles, as well as when cells are under stress. This large concentration range yet unseen for other physiological substances raise a great challenge to the optical bioimaging community both intellectually and in practical sense. The significance of the proposed research lies in the fact that if successful, it will provide valuable tools for quantitative analysis of Zn2+ over its complete physiological concentration range in all cell types. Therefore, the proposed studies will facilitate the elucidation of Zn2+ homeostatic pathways and to identify therapeutic targets for developing diagnosis and treatment of diseases whose pathology is related to deviation of Zn2+ homeostasis. The innovation of proposed research is reflected in our rationally designed heteroditopic platform that translates three coordination states (non-, mono-, and di-coordinated) to three fluorescence states (non- fluorescent, fluorescent at one color, and fluorescent at a different color), thus providing a convenient analytical protocol for quantitative Zn2+ analysis over a large concentration range. In this grant application, we propose strategies for developing fluorescent probes for physiological Zn2+ with large effective concentration ranges that will be used in quantitative live-cell imaging of free Zn2+. Built upon a heteroditopic arylvinyl-bipy system developed in our laboratory, we will specifically address the issues stipulated in the Specific Aims: (1) design and preparation of new probe molecules to increase the sensitivity of Zn2+ quantification (while maintaining a large effective concentration range); (2) incorporation of known laser dye structures into our unique heteroditopic framework to produce probes for live-cell fluorescence microscopic applications; (3) development of a new heteroditopic framework whose emission profile is insensitive to solvent polarity; and (4) applications of our probes in biological imaging, in particular in hippocampal neurons, using fluorescence microscopy.